RESUMO
Phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] is a key regulator of cell signaling that acts by recruiting proteins to the cell membrane, such as at the leading edge during cell migration. Here, we show that PtdIns (3,4,5)P3 plays a central role in filopodia formation via the binding of myosin-X (Myo10), a potent promoter of filopodia. We found that the second pleckstrin homology domain (Myo10-PH2) of Myo10 specifically binds to PtdIns(3,4,5)P3, and that disruption of this binding led to impairment of filopodia and partial re-localization of Myo10 to microtubule-associated Rab7-positive endosomal vesicles. Given that the localization of Myo10 was dynamically restored to filopodia upon reinstatement of PtdIns(3,4,5)P3-binding, our results indicate that PtdIns(3,4,5)P3 binding to the Myo10-PH2 domain is involved in Myo10 trafficking and regulation of filopodia dynamics.
Assuntos
Miosinas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Pseudópodes/metabolismo , Animais , Células COS , Chlorocebus aethiops , Endossomos/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Ligação Proteica , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologiaRESUMO
The NaChBac sodium channel from Bacillus halodurans is a homologue of eukaryotic voltage-gated sodium channels. It can be solubilized in a range of detergents and consists of four identical subunits assembled as a tetramer. Sodium channels are relatively flexible molecules, adopting different conformations in their closed, open and inactivated states. This study aimed to design and construct a mutant version of the NaChBac protein that would insert into membranes and retain its folded conformation, but which would have enhanced stability when subjected to thermal stress. Modelling studies suggested a G219S mutant would have decreased conformational flexibility due to the removal of the glycine hinge around the proposed gating region, thereby imparting increased resistance to unfolding. The mutant expressed in Escherichia coli and purified in the detergent dodecyl maltoside was compared to wildtype NaChBac prepared in a similar manner. The mutant was incorporated into the membrane fraction and had a nearly identical secondary structure to the wildtype protein. When the thermal unfolding of the G219S mutant was examined by circular dichroism spectroscopy, it was shown to not only have a Tm approximately 10 degrees C higher than the wildtype, but also in its unfolded state it retained more ordered helical structure than did the wildtype protein. Hence the G219S mutant was shown to be, as designed, more thermally stable.
Assuntos
Proteínas de Bactérias/química , Escherichia coli/metabolismo , Canais de Sódio/química , Bacillus/química , Bacillus/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Dicroísmo Circular , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/biossíntese , Proteínas Mutantes/química , Conformação Proteica , Desnaturação Proteica , Estabilidade Proteica , Alinhamento de Sequência , Canais de Sódio/biossíntese , Canais de Sódio/genéticaRESUMO
NaChBac from Bacillus halodurans is a bacterial homologue of mammalian voltage-gated sodium channels. It has been proposed that a NaChBac monomer corresponds to a single domain of the mammalian sodium channel and that, like potassium channels, four monomers form a tetrameric channel. However, to date, although NaChBac has been well-characterized for functional properties by electrophysiological measurements on protein expressed in tissue culture, little information about its structural properties exists because of the difficulties in expressing the protein in large quantities. In this study, we present studies on the overexpression of NaChBac in Escherichia coli, purification of the functional detergent-solubilized channel, its identification as a tetramer, and characterization of its secondary structure, drug binding, and thermal stability. These studies are correlated with a model produced for the protein and provide new insights into the structure-function relationships of this sodium channel.
Assuntos
Proteínas de Bactérias/química , Canais de Sódio/química , Bacillus/genética , Bacillus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Western Blotting , Dicroísmo Circular , Dimerização , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Biológicos , Ligação Proteica , Estrutura Secundária de Proteína , Canais de Sódio/genética , Canais de Sódio/metabolismoRESUMO
Receptor priming of low-pH-triggered virus entry has been described for an enveloped virus (15). Here we show with major group human rhinoviruses (HRV) and its intercellular adhesion molecule-1 receptor that nonenveloped viruses follow this novel cell entry principle. In vitro the receptor primed HRV for efficient uncoating at mild low pH (5.5 to 6.0). Agents preventing endosomal acidification reduced or blocked rhinovirus cell infection, while nocodazole had no effect on infection of any serotype tested. The entry inhibitory effect of lysosomotropic agents was overcome by exposing cell-internalized HRV to mild low pH (5.5 to 6.0). We therefore conclude that receptor priming of major group HRV must occur in vivo as well. Cooperation of a cellular receptor and low pH in virus uncoating will polarize the exit of the genome to the receptor-bound, membrane-proximal region of the virus particle during acidification of endosomes. This process must be required for efficient penetration of the cellular membrane by viruses.